The Preparation Of Cucurmosin-2 And The Study Of Its Mechanism Inducing Apoptosis Of HepG2 Cancer Cell

Object:The aim of this study is to obtain a novel ribosome inactivating protein—Cucurmosin 2(CUS2), to explore its activity of inhibition on human liver cancer cells(HepG2,ect.)in vitro.And,to study the possible molecular mechanism inducing apoptosis of HepG2 cancer cell which can provide the experimental and theoretical basis of potential application into liver cancer therapy. Methods:1. The Sp-Sepharose Fast Flow cation exchange chromatography was used to purify CUS2. SDS-PAGE polyacrylamide gel electrophoresis was applied to identify CUS2 and calculate its molecular weight.BCA method was used to mesure the concentration of CUS2.2. SRB method was used to detect the effect of CUS2 in vitro on proliferation of the human HepG2 cells and human embryo liver cell line L-02 cell, and evaluate the selective effect of CUS2 on human liver cancer cells.3. Hoechst 33258 fluorescent staining was used to analyze the effect of CUS2 on HepG2 cell.4. Electron microscopy was used to observe the changes of HepG2 cells after CUS2 intervence.5. PI flow cytometry was used to detect the effects of CUS2 on HepG2 cell proliferation cycle.6. ELISA was used to detect the enzyme activity of Caspase 3, Caspase 9 in HepG2 cells after CUS2 intervence.7. Western blot was used to detect the protein expression level of golgi phosphoprotein-3(GOLPH3), mTOR, Akt, Raf, Ras, Caspase 3 in HepG2 cell after CUS2 intervence. Results:1. 280 nm protein nucleic acid detector detected two test elution peak and obtained two pumpkin proteins—CUS1 and CUS2, through Sp-Sepharose Fast Flow cation exchange chromatography column chromatography. CUS1 and CUS2 identified by SDS-PAGE reached the electrophoresis pure.Their molecular weights were about 28.2 k Da and 29.0 k Da, respectively. The concentration of CUS2 was 5.26 mg/ml.2. The results of SRB showed that significantl inhibition of CUS2 on HepG2 cells in vitro,which was cultured in a dose-and-time-depedent manner. The rate of CUS2 proliferation inhibition on HepG2 cells at different concentrations(0.625, 1.25, 2.50, 5.00, 10.00, 20.00, and 5.00 ?g/m L) after 72 h were 9.50%, 15.74%, 27.71%, 15.74%, 55.70%, 69.10% and 55.70%, respectively.The IC50 of CUS2 was(6.74±0.25)?g/m L. The IC50 of CUS2 on HepG2 cells in 24, 48,72,96 and 120 h were(38.23±1.83),(17.97±1.93),(7.28± 0.26),(6.27±0.28) and(5.50±0.37) ?g/m L, respectively. The IC50 of CUS2 on human embryo liver L-02 cells was about(34.29±3.09) ?g/m L. The results preliminary showed that CUS2 had obvious destruction on human liver cancer cells HepG2, which was far more effective than those of the normal liver tissue.3. Fluorescence microscopy obversed typical apoptotic morphological characteristics of human liver HepG2 cells after CUS2 intervence: integral cell membrane, smaller volume, agglutination of nuclear chromatin and karyopyknosis. Electron microscope showed that the nuclear chromatin was agglutinated, part of the nuclear membrane was disappeared, or saw a nuclear fragmentation and apoptotic body in some apoptotic cells. Flow cytometry showed that the apoptosis rate of CUS2 at 0, 2.50, 10.00 and 2.50 ?g/m L) after 72 h were(2.37 ±0.19) %,(17.22 ±0.29) %,(36.78 ±1.81) % and(66.35± 2.35) %, respectively. Compared with the control group, CUS2 mainly blocked HepG2 cells in S phase and G2 phase, thus induced cell apoptosis. The inductiong apoptosis rate was increased gradually in a dose-depedent manner.4. ELISA showed Caspase 3 and Caspase 9 in HepG2 cells were activated by CUS2 at 0, 2.50, 10.00 and 2.50 ?g/m L after 72 h in a dose-depedent manner. Western blot showed that GOLPH3, mTOR, Akt, Raf and Ras proteins in HepG2 cells were weakened, following by increasing concentration of CUS2. However, active fragments of caspase 3 was activated.The role of CUS2 was dose-depedent. Conclusions:1. CUS2 was a kind of novel ribosome inactivating proteins, which was different from CUS1. The molecular weight of CUS2 was about 29.0 k Da.2. 2. CUS2 effectively induced the apoptosis of liver cancer cell HepG2.3. The molecular mechanism of cell apoptosis of HepG2 cells by CUS2 may be correlated with the downregulation of CUS2 on the expression levels of GOLPH3, mTOR, Akt, Raf, Ras proteins and its activation of caspase 3 following by caspase 9.